Curcumin increases gelatinase activity in human neutrophils by a p38 mitogen-activated protein kinase (MAPK)-independent mechanism

Abstract

Curcumin has been found to possess anti-inflammatory activities and neutrophils, key players in inflammation, were previously found to be important targets to curcumin in a few studies. For example, curcumin was found to induce apoptosis in neutrophils by a p38 mitogen-activated protein kinase (MAPK)-dependent mechanism. However, the role of curcumin on the biology of neutrophils is still poorly defined. To study the role of curcumin on neutrophil degranulation and to determine the role of p38 MAPK, human neutrophils were freshly isolated from healthy individuals and incubated in vitro with curcumin. Degranulation was studied at three levels: surface expression of granule markers by flow cytometry; release of matrix metallopeptidase-9 (MMP-9 or gelatinase B) enzyme into supernatants by Western blot; and gelatinase B activity by zymography. Activation of p38 MAPK was studied by monitoring its tyrosine phosphorylation levels by western blot and its role by the utilization of a pharmacological inhibitor. The results indicate that curcumin increased the cell surface expression of CD35 (secretory vesicle), CD63 (azurophilic granules), and CD66b (gelatinase granules) in neutrophils. Also, curcumin increased the release and enzymatic activity of gelatinase B in the extracellular milieu and activated p38 MAP kinase in these cells. However, in contrast to fMLP, curcumin-induced enzymatic activity and secretion of gelatinase B were not reversed by use of a p38 inhibitor. Finally, it was found that curcumin was able to enhance phagocytosis. Taken together, the results here demonstrate that curcumin induced degranulation in human neutrophils and that the increased gelatinase activity is not dependent on p38 MAPK activation. Therefore, degranulation is another human neutrophil function that could be modulated by curcumin, as well as phagocytosis.     

SOCS3 dictates the transition of divergent time-phased events in granulocyte TNF-a signaling

Tumor-necrosis factor-a （TNF-a）-driven nuclear factor-KB （NF-KB） activation and apoptosis are opposing pathways; the growing recognition of these conflicting roles of TNF-a is perplexing. Here, we show that inflammation and apoptosis are time-phased events following TNF-a signaling and that emergence of suppressor of cytokine signaling 3 （SOCS3） expression limits the ongoing NF-KB activation and promotes apoptosis; further, we suggest an altered view of how inflammatory diseases are initiated and sustained. In vitro, TNF-a （50 ng/ml） induced granulocyte SOCS3 protein, inhibited nuclear accumulation of the p65NF-KB subunit and enhanced apoptosis, as shown by DNA laddering, annexin V positivity, and overexpression of caspase-3 and Bax in the late phase, whereas the early phase was marked by NF-KB activation. Conversely, SOCS3 knockdown by small interfering RNA （siRNA） inhibited granulocyte apoptosis and enhanced nuclear accumulation of p65 and 5＇ lipooxygenase expression in the late phase of TNF-a signaling. As apoptosis is associated with SOCS3 abundance, we suggest that these divergent TNF-a-driven events are time-phased, interconnected, opposing control mechanisms and one of the central features through which the immune system resolves pulmonary inflammation. Dysregulation may initiate mucosal inflammation, thus changing the landscape of asthma theraov.     

Evaluation of myeloid and lymphoid dendritic cells in peritoneal fluid in women with non-malignant ovarian tumors

PROBLEM: Identification of myeloid and lymphoid dendritic cells (DCs) in peritoneal fluid (PF) and peripheral blood (PB) of patients with ovarian pathology. METHOD OF STUDY: PF and PB were collected from 60 patients who underwent laparoscopy because of non-malignant ovarian tumors. Mononuclear cells were separated by gradient centrifugation. The cell surface antigens were determined by flow cytometry using monoclonal antibodies. RESULTS: Both myeloid and lymphoid DCs were detected in PF and PB of women with ovarian tumors. The percentage of myeloid DCs was significantly higher in PF than in PB. The concentration of PF myeloid DCs was the highest (P < 0.05) in patients with dermoid cysts (0.67 x 10(6)/mL PF) in comparison with the other studied groups, excluding patients with normal pelvis. CONCLUSIONS: Domination of myeloid and not lymphoid cells in PF may support the hypothesis that local PF immune disturbances may play a role in some non-malignant ovarian pathology.     

Spontaneous death of bone marrow and peripheral blood cells during remission of acute lymphoblastic leukemia in children

Programmed antileukemic chemotherapy is associated with prolonged cytotoxic effects on not only tumor cells, but on intact bone marrow and peripheral blood cells as well. The maximum spontaneous cell death is observed immediately after the end of therapy. It decreases with time after therapy, but does not reach the level observed in healthy subjects and does not depend on the type of therapy. The percentage of dead cells in peripheral blood directly correlates with the percentage of dead cells in the bone marrow. The processes of cell death in the peripheral blood and bone marrow are synchronous and parallel.     

Flow Cytometry Analysis of Lymphocyte Subpopulations in Peritoneal Fluid of Women With Endometriosis

PROBLEM: We investigated the lymphocyte subpopulations in peripheral blood (PB) and peritoneal fluid (PF) of women with and without endometriosis to evaluate if the decreased natural killer (NK)-mediated cytotoxicity in women with endometriosis was due to a quantitative defect or not. METHOD: The PB and PF mononuclear cells of 59 women undergoing a diagnostic laparoscopy for pain and/or infertility were analyzed by flow cytometry. RESULTS: The number and concentration of PF mononuclear cells (MC) was increased in women with endometriosis compared to women without endometriosis. The monocyte/macrophage marker (CD14) was expressed on 70.3 and 66.9% of PFMC of women with and without endometriosis, respectively. The CD4/CD8 ratio was inverted in the PF, and this was more pronounced in women with endometriosis. In the PF of women with endometriosis, 41.3% of the lymphocytes were CD8 positive, compared to 34.3% in women without endometriosis. The percentage of NK positive lymphocytes in PF, using three different monoclonal antibodies directed against NK cell markers (CD57, CD 16, and CD56) were not different between women with and without endometriosis. In women with endometriosis, 12.7,9.5, and 28.8% of lymphocytes were CD57, CD16, and CD56 positive, respectively. CONCLUSION: PFMC consisted mainly of phagocytic and human leukocyte antigen (HLA)-restricted or HLA unrestricted cytotoxic cells capable of reacting to various antigens entering the cavity from the lower genital tractus. Furthermore, the decreased NK activity reported in PB and PF of women with endometriosis was not likely to be caused by a quantitative defect, since the percentage of NK positive lymphocytes was not different between women with and without endometriosis.     

G-CSF对外周血树突状细胞亚群比例的影响

目的 :了解G CSF对小鼠外周血树突状细胞 (DC)亚群比例的影响。方法 :以不同剂量 (0、5 .0、10 .0或 15 .0 μg)的G CSF ,分别皮下注射于BALB/c小鼠 ,连续 6d ,每天 1次。第 6天分离外周血DC ,用流式细胞仪检测DC1(CD11c+ CD8a-)和DC2 (CD11c+ CD8a+ )的比例并计算其绝对值。结果 :经不同剂量的G CSF刺激后 ,外周血DC1的绝对值无显著变化 ,而DC2的绝对值增加 5倍 (9.6× 10 6/Lvs 5 5 .1× 10 6/L ,P <0 .0 1) ,DC1/DC2比值降低 (4.2vs 0 .7,P <0 .0 1)。结论 :G CSF在体内可提高外周血DC2的绝对数 ,对DC1的数无明显影响 ,从而使DC1/DC2的比例倒置     

Study of morphology and rigidity of neutrophilic granulocyte membrane in the real time mode by scanning probe microscopy

The rigidity of neutrophilic granulocyte membrane was determined: 2.1±0.7 kPa. Scanning in the contact mode did not modify cell morphology, while spectroscopy caused nonspecific swelling reaction and a 3-fold increase in cell volume. Spectroscopy had no effect on rigidity of neutrophilic granulocyte membrane. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 141, No. 6, pp. 712–714, June, 2006     

Adjuvant treatment increases the resistance to Mycobacterium avium infection of mycobacteria-susceptible BALB/c mice.

Systemic lupus erythematosus (SLE)-like anti-IgG Fab autoantibodies (autoAb) were induced in rabbits by immunization with either human, mouse or rabbit anti-DNA Ab. In direct-binding radioimmunoassay (RIA), affinity-purified anti-normal rabbit (NR) Fab autoAb cross-reacted with normal mouse (NM) Fab, ssDNA (but not dsDNA), poly(dA,dT), and RNA polymerase I (RPI). Affinity-purified anti-NM IgG Ab isolated from the same antisera cross-reacted with NR Fab, ssDNA and RPI. In inhibition RIA, soluble NR Fab inhibited anti-NR Fab binding to NR Fab and ssDNA, but enhanced binding to RPI. In contrast, ssDNA or RPI inhibitors had no effect upon autoAb binding to NR Fab. Anti-DNA, anti-RPI and anti-RPI 190 kD subunit autoAb, induced by immunization with lupus mouse anti-DNA Ab, also reacted with NM Fab, but were idiotypically specific for lupus mouse anti-DNA Fab. Further, rabbit anti-DNA and anti-RPI IgG autoAb, induced by immunization with rabbit anti-DNA IgG, were each idiotypically specific for homologous and autologous rabbit anti-ssDNA Fab. Together, these data provide evidence that anti-DNA, anti-RPI and anti-Fab autoAb are linked in a complex, multiple-specific and perhaps regulatory, immune response idiotype network in SLE.     

Follicular lymphoma transformed to “double‐hit” B lymphoblastic lymphoma presenting in the peritoneal fluid

Lymphomas showing both MYC/8q24 rearrangement and IGH@BCL2/t(14;18)(q32;q21), also referred to as “double‐hit” or “dual‐hit” lymphomas (DHL) are rare B‐cell malignancies with a germinal center B‐cell immunophenotype and heterogeneous cytologic and histologic features. Such lymphomas may arise de novo or through transformation of follicular lymphomas and are classified either as “B‐cell lymphoma, unclassifiable, with features intermediate between diffuse large B‐cell lymphoma (DLBCL) and Burkitt lymphoma (BL)” (most commonly), DLBCL, or, rarely, as B‐lymphoblastic lymphoma. We report a case of B‐lymphobastic lymphoma arising through transformation of follicular lymphoma diagnosed on peritoneal fluid cytology, flow cytometry, and cytogenetic studies in a 53‐year‐old man who presented with abdominal pain, shortness of breath, night sweats, extensive lymphadenopathy, pleural effusion, and ascites. Cytologic examination of the ascitic fluid showed two distinct populations of neoplastic lymphoid cells, a predominant population of larger cells with fine powdery (“blastic”) chromatin, visible to prominent nucleoli and occasional small cytoplasmic vacuoles and a less numerous population of smaller cells with centrocytic morphology. Flow cytometry also showed two distinct monotypic B‐cell populations, both expressing CD10, and TdT‐positivity was demonstrated immunohistochemically. Fluorescence in situ hybridization (FISH) demonstrated both MYC rearrangement and IGH/BCL2 gene fusion and cytogenetic analysis showed a complex karyotype including both t(14;18)(q32;q21) and t(8;22)(q24.1;q11.2). Since DHL pursue an aggressive clinical course, respond poorly to therapy, and have a poor outcome, it is important to suspect the diagnosis when encountering neoplastic lymphoid cells that are difficult to classify in effusion cytology specimens and to order the appropriate immunophenotyping and cytogenetic studies. Diagn. Cytopathol. 2013;41:986–990. © 2012 Wiley Periodicals, Inc.     

Delayed effect of antileukemic polychemotherapy on cell death and proliferation during remission of acute lymphoblastic leukemia in children

The effect of programmed antileukemic drug therapy on cells of the tumor clone is not selective, and the drugs affect also intact bone marrow and peripheral blood cells. The cytotoxic effect of antitumor therapy is attained through triggering apoptosis and does not depend on the type of drug therapy. The treatment induces long-lasting changes in spontaneous cell death processes. The disturbances in the kinetics of cell death caused by drug therapy are compensated by changes in proliferative activity of bone marrow cells.     

Intracellular cytokine profile of T lymphocytes in patients with chronic obstructive pulmonary disease

Chronic obstructive pulmonary disease (COPD) is characterized by an excessive inflammatory response to inhaled particles, mainly tobacco smoking. T lymphocytes are important regulatory cells that secrete several cytokines and participate actively in this inflammatory response. According to the pattern of cytokines secreted, the immune response is classified as cytotoxic or type 1 [interferon (IFN)-gamma-, interleukin (IL)-2-dependent] and humoral or type 2 (IL-4-, IL-5-, IL-10- and IL-13-dependent). This paper sought to compare the intracellular profile of cytokine expression determined by flow cytometry in T lymphocytes harvested from bronchoalveolar lavage (BAL) and peripheral blood in patients with COPD, smokers with normal lung function and never smokers. We found that BAL T lymphocytes from COPD patients had a higher percentage of positive stained cells for most of the cytokines analysed when compared to never smokers or smokers with normal lung function. Differences reached statistical significance for IL-4, IL-10 and IL-13, particularly in CD8(+) T cells. Furthermore, the expression of most of these cytokines was related inversely to the degree of airflow obstruction present suggesting local activation and/or selective homing of T lymphocytes to the lungs in COPD patients. These observations were not reproduced in circulating T lymphocytes. These results suggest that BAL T lymphocytes in patients with COPD produce more cytokines than in controls and tend to show a type 2 pattern of intracellular cytokine expression, particularly a Tc-2 profile. This is related inversely to the degree of airflow obstruction present.     

A lesson from the wild: The natural state of eosinophils is Ly6Ghi

With a long history of promoting pathological inflammation, eosinophils are now emerging as important regulatory cells. Yet, findings from controlled laboratory experiments so far lack translation to animals, including humans, in their natural environment. In order to appreciate the breadth of eosinophil phenotype under non‐laboratory, uncontrolled conditions, we exploit a free‐living population of the model organism Mus musculus domesticus. Eosinophils were present at significantly higher proportions in the spleen and bone marrow of wild mice compared with laboratory mice. Strikingly, the majority of eosinophils of wild mice exhibited a unique Ly6G^hi phenotype seldom described in laboratory literature. Ly6G expression correlated with activation status in spleen and bone marrow, but not peritoneal exudate cells, and is therefore likely not an activation marker per se. Intermediate Ly6G expression was transiently induced in a small proportion of eosinophils from C57BL/6 laboratory mice during acute infection with the whipworm Trichuris muris, but not during low‐dose chronic infection, which better represents parasite exposure in the wild. We conclude that the natural state of the eosinophil is not adequately reflected in the standard laboratory mouse, which compromises our attempts to dissect their functional relevance. Our findings emphasize the importance of studying the immune system in its natural context – alongside more mechanistic laboratory experiments – in order to capture the entirety of immune phenotypes and functions.     

cAIMP administration in humanized mice induces a chimerization-level-dependent STING response

It is well understood that the STING signalling pathway is critical for generating a robust innate immune response to pathogens. Human and mouse STING signalling pathways are not identical, however. For example, mice lack IFI16, which has been proven important for the human STING pathway. Therefore, we investigated whether humanized mice are an appropriate experimental platform for exploring the human STING signalling cascade in vivo. We found that NOG mice reconstituted with human cord blood haematopoietic stem cells (humanized NOG mice) exhibit human STING signalling responses to an analogue of the cyclic di-nucleotide cGAMP. There was an increase in the proportions of monocytes in the lungs of mice receiving cGAMP analogue. The most robust levels of STING expression and STING-induced responses were observed in mice exhibiting the highest levels of human chimerization. Notably, differential levels of STING in lung versus spleen following cGAMP analogue treatment suggest that there are tissue-specific kinetics of STING activation and/or degradation in effector versus inductive sites. We also examined the mouse innate immune response to cGAMP analogue treatment. We detected that mouse cells in the immunodeficient NOG mice responded to the cGAMP analogue and they do so with distinct kinetics from the human response. In conclusion, humanized NOG mice represent a valuable experimental model for examining in vivo human STING responses.     

High dimensional analysis reveals distinct NK cell subsets but conserved response to stimulation in umbilical cord blood and adult peripheral blood

Growing interest surrounds adoptive cellular therapies utilizing Natural Killer (NK) cells, which can be obtained from various sources, including umbilical cord blood (UCB) and adult peripheral blood (APB). Understanding NK cell receptor expression and diversity in such cellular sources will guide future therapeutic designs. We used a 20-color flow cytometry panel to compare unstimulated and cytokine-activated UCB and APB NK cells. Our analysis showed that UCB NK cells express slightly higher levels of the immune checkpoints PD-1, TIGIT, and CD96 compared to their APB counterparts. Unsupervised hierarchical clustering and dimensionality reduction analyses revealed enrichment in CD56^neg as well as mature NKp46^neg and CD56⁺CD16⁺ NK cell populations in UCB whereas CD57⁺ terminally differentiated NK cells with variable expression of KIRs and CD16 were found in APB. These populations were conserved following stimulation with IL-12, IL-15, and IL-18. Cytokine stimulation was associated with the downregulation of TIGIT and CD16 on multiple NK cell subsets in UCB and APB. Among UCB CD16⁻ NK cell populations, TIGIT⁺ NK cells produced more IFN-γ than their TIGIT⁻ counterparts. Our data demonstrate higher immune checkpoint expression on UCB NK cells compared to APB. However, the expression of TIGIT immune checkpoint is not indicative of NK cell exhaustion.     

Neonatal aldosterone administration alters later response to adrenalectomy and aldosterone infusion in the rat

Juvenile and adult infusions of mineralocorticoids promote weight gains in rats. However, nothing is known about the effects of very early administration. As such treatments often bring about enduring changes, we examined the impact of neonatal (days 1-5 of life), followed by early adult (days 45-57 of life), administration of aldosterone (ALDO) on the weight gains of free-fed rats. Analysis revealed that neonatal ALDO exposure altered the pattern of weight gain during the later period. Specifically, acute neonatal exposure increased weight gain in adrenalectomized young adult rats. At the same time, it appeared to reduce the effects of chronic ALDO exposure in these animals.     

Inhibition of the neurogenic inflammatory response by lidocaine in rat skin

Axon reflex vasodilatation and neurogenic plasma extravasation are characteristic cutaneous vascular responses mediated by neuropeptides released from stimulated capsaicin-sensitive sensory nerve endings. Intracutaneous injections of local anaesthetics inhibit the axon-reflex flare elicited by chemical irritants in human skin. Results of earlier reports on the effects of local anaesthetics on neurogenic plasma extravasation are controversial. The aim of the present study, therefore, was to re-examine the effect of the local anaesthetic lidocaine on the neurogenic inflammatory response of rat skin. The effects of lidocaine on cutaneous inflammatory reactions were measured quantitatively by means of the Evans blue technique. Intracutaneous injection of lidocaine resulted in a dose-dependent inhibition of the neurogenic inflammation elicited by mustard oil and of the dye leakage response to compound 48/80 or histamine. It is suggested that the site of this inhibition is beyond the sensory nerve terminal, presumably at the level of the vascular endothelium.accepted by K. Brune     

Short-term Morphological response of the rat testis to administration of five chemotherapeutic agents

As cancer survival rates improve, there is increasing concern about the adverse effects of chemotherapeutic agents on male fertility. Five chemotherapeutic agents (amethop-terin, AP or methotrexate; doxorubicin, DX; cytoxan or cyclophosphamide, CP; cisplatinum, CDDP; and 5-fluorouracil, 5-FU) which belong to three different categories of chemotherapeutic agents (antimetabolite, antibiotic, alkylating agent, alkylating agent, antimetabolite, respectively) were given systemically to adult rats to determine the short-term morphological patterns of response in the testis, and the testes were examined by light microscopy. Morphological patterns of response were found to be highly characteristic for each agent, and some shared morphological responses were evident. All except one chemotherapeutic agent (5-FU) caused spermatogonial damage. Among the defects seen were probable degenerating meiotic spermatocytes (CDDP), presence of micronuclei (DX), “arrested” spermatid development (5-FU), and abnormally shaped step 15 spermatids (5-FU). Damage that could be due to the effect of an agent on the Sertoli cell was failure of sperm release (5-FU, CDDP, DX, and AP), increase in the Sertoli cell lipid (5-FU), and malorientation of step 8 spermatids (5-FU, DX). The varied patterns of damage observed are a possible explanation of why the reproductive recovery potential in cancer patients undergoing chemotherapy is variable and drug-specific.     

Endogenous opioids and the first suckling episode in the rat

Endogenous activity at opioid receptors affects the appetitive behavior of Caesarean-delivered rat pups during presentation of a surrogate nipple that provides milk. Blockade of opioid receptors by peripheral injection of naloxone has no effect on responses evoked by the surrogate nipple. Similarly, blockade of caudal brain opioid receptors by injection of naloxone into the cisterna magna has no effect on the pup's behavior in response to the surrogate nipple. However, blockade of rostral opioid receptors by injection of naloxone into the cerebral ventricles increases the latency to the first oral grasp response, decreases total time on the nipple, and virtually eliminates ingestion of milk from the surrogate nipple (Experiment 1). Blockade of endogenous opioid activity does not affect responses to a nipple that provides distilled water (Experiment 2) or to an empty surrogate nipple (Experiment 3). These data indicate that during the initial suckling episode endogenous opioids in rostral brain regions affect the pup's behavioral responses to the nipple. The results are consistent with the hypothesis that milk engages opioid systems during the first suckling and that endogenous opioids play a role in early suckling. © 1998 John Wiley & Sons, Inc. Dev Psychobiol 33: 175–183, 1998     

Heterogeneity of brainstem blood flow response to hypoxia in the anesthetized rat

Cerebral blood flow is strictly regulated during hypoxic stress. Because of the preponderant role of the brainstem in cardiorespiratory controls, blood flow response to hypoxia is stronger in this region than in the cortex. However, the brainstem is made up of various regions which differ in their responsiveness to chemical stimuli. The objective of this study was to evaluate the distribution of blood flow during hypoxia using microsphere deposition methods in three brainstem regions containing key structures in cardiorespiratory controls: the nucleus tractus solitarus (NTS), the ventral respiratory groups (VRG) and the pontine respiratory groups (PRG). Microsphere injections were made during normoxia (FIO2=0.21) and after 15 min of hypoxia (FIO2=0.21). Based on this index, blood flow increase during hypoxia was higher in the VRG than in the dorsal part of the brainstem, containing the NTS and the PRG (P=0.002, n=10). These results suggest that blood flow response to hypoxia favours O(2) delivery in brainstem regions involved in respiratory rhythm generation.